Transgenic animals

ABSTRACT

There is discloses a transgenic mouse over-expressing a potassium channel BEC1, which can be used as an effective tool for screening a substance for antidementia or a substance to improve learning and memory.  
     Also disclosed are an in vivo screening method of a substance for antidementia or a substance to improve learning and memory, which uses the learning and memory abilities of said mouse as the index, and an in vivo screening of a substance for antianxiety, which uses acceleration of anxiety as the index.  
     In addition, there is disclosed a method for producing a pharmaceutical composition for antidementia, learning and memory improvement use or antianxiety, using a substance capable of inhibiting the learning and memory potassium channel activity as the active ingredient which can be obtained by the aforementioned screening method of the present invention.

TECHNICAL FIELD

[0001] This invention relates to a model animal to be used as aneffective tool for screening a substance for antidementia, a substanceto improve learning and memory or a substance for antianxiety, and amethod for screening a novel substance for antidementia, substance toimprove learning and memory or substance for antianxiety, which usessaid model animal.

BACKGROUND OF THE INVENTION

[0002] Studies on the disorders in learning new information whichoccupies an important part of the mental activities of human and on thedisorders in keeping and recalling already learned information are mainthemes for elucidating higher brain functions which have been regardedas the final frontier of life science. However, the cause for thedisorders is unclear even at present and there are no sufficientlyeffective medicaments yet, so that early development of therapeuticdrugs is greatly demanded. Even in the case of medicaments which havebeen reported to be effective in improving impairment in learning andmemory of dementia patients to a certain degree, it has been pointed outthat the duration of action is short and the clinically effective rangeis narrow (Feldman H. et al., (2001), Neurology, 57 (4), 613 - 20), thatside effects such as hepatic toxicity and the like are generated(Summers W. K. et al., (1989), Lancet, 1 (8640), 729) and the like. Inaddition, for the purpose of finding an agent showing a usefultherapeutic effect, it is necessary to take into consideration not onlyits in vitro efficacy but also in vivo bioavailability and transferringactivity into the brain of the agent itself. Accordingly, the presenceof a model animal which can be analyzed at individual level is essentialfor the elucidation of onset mechanism of the disease, prevention ofonset of the disease or improvement of its pathologic condition,development of medicinal techniques for the treatment and ofmedicaments, and the like.

[0003] The transgenic mouse is expected as a method for displayingfunctions of genes in the living body for development of models forrespective human diseases and development of various therapeutic drugsusing these models. Including the c-myc oncogene-introduced transgenicmouse as a leukemia model which received a patent for a living life forthe first time in the world (U.S. Pat. No. 5,087,571), variouspathological model mice have been introduced, such as a prostatichyperplasia mouse (U.S. Pat. No. 5,175,383), a diabetesmellitus-generating mouse (Japanese Patent 2771493), an Alzheimer'Sdisease model mouse (Games D. et al., (1995), Nature, 373 (6514), 523-7)and the like. Although many of the dementia-related transgenic animalsrepresented by an APP transgenic show pathologic changes of dementia, itis difficult to detect impairment of learning and memory as a behavioralpattern in these animals, so that attempts have been made detect it byadding some factors such as aging and the like (Moran P. M. et al.,(1995), Proc. Natl. Acad. Sci. USA, 92, 5341-5345; Hsiao K. et al.,(1996), Science, 274, 99-102; Arendash G. W. et al., (2001), Brain Res.,891, 42-53). However, because preparation of such transgenic animalstakes time, it was not practical to conveniently mass-prepare transgenicanimals capable of detecting disorders of learning and memory as abehavioral pattern for use in the screening of a substance forantidementia.

[0004] Accordingly, demands have been directed toward a model animal tobe used as an effective tool for screening a substance for antidementiaor a substance to improve learning and memory, and a method forscreening a substance for antidementia or a substance to improvelearning and memory, which uses said model animal.

[0005] On the other hand, anxiety is a physiological phenomenonfunctioning as a warning against a present or potential danger. Anxietybecomes pathologic when it occurs without a real danger or its emotionalstrength intensifies. It is known that both of the physiological andpathological anxieties can threaten life when an organ disorder isgenerated in spite of its previous presence and may cause or permanentlykeep various physiological dysfunctions. There are various reports ontransgenic animals as models of anxiety (Holmes A., (2001), Neurosci.Biobehav. Rev., 25 (3), 261-73). However, there is no applicable andoptimum agent having less side effects even now.

[0006] Regarding a potassium channel BEC1 which can be used in preparingthe transgenic animal of the present invention, the present inventorshave disclosed it in WO 99/37677, and there are many reports regardingpolynucleotides and polypeptides having a homology of from 98 to 99%with the BEC1 (WO 00/05346, WO 00/09534, WO 99/43696). However, a BEC1transgenic animal was not prepared in reality, and its functions werenot necessarily understood completely.

DISCLOSURE OF THE INVENTION

[0007] As a result of extensive studies, the present inventors havesucceeded in preparing a transgenic mouse over-expressing the potassiumchannel BEC1 (Miyake A. et al., (1999), J. Biol. Chem., 274,25018-25025) and found that, since the learning and memory abilities ofsaid transgenic mouse is reduced, it can be used as a tool for screeninga substance for antidementia or a substance to improve learning andmemory. Also, it was found unexpectedly that, since anxiety of saidtransgenic mouse is accelerated, it can be used as a tool for screeninga substance for antianxiety. In addition, an in vivo screening systemfor a substance for antidementia or a substance to improve learning andmemory was constructed using the learning and memory abilities of saidtransgenic mouse as the index, and an in vivo screening system for asubstance for antianxiety using the anxiety of said transgenic mouse asthe index. As the results, the present invention was accomplished byproviding a BEC1 transgenic animal as a model animal to be used as aneffective tool for screening a substance for antidementia, a substanceto improve learning and memory or a substance for antianxiety, and amethod for screening a substance for antidementia, a substance toimprove learning and memory or a substance for antianxiety, which usessaid transgenic animal.

[0008] Accordingly, the present invention relates to,

[0009] (1) a transgenic animal introduced with a sequence comprising apromoter and a polynucleotide encoding a potassium channel which is apolypeptide consisting of the amino acid sequence represented by SEQ IDNO:2 or a polypeptide comprising an amino acid sequence in which from 1to 10 of amino acids in the amino acid sequence represented by SEQ IDNO:2 are deleted, substituted and/or added,

[0010] (2) a transgenic animal which is a non-human animal or aoffspring animal thereof obtained by effecting ontogenesis of atotipotent cell introduced, together with a promoter, with apolynucleotide encoding a potassium channel which is a polypeptideconsisting of the amino acid sequence represented by SEQ ID NO:2 or apolypeptide comprising an amino acid sequence in which from 1 to 10 ofamino acids in the amino acid sequence represented by SEQ ID NO:2 aredeleted substituted and/or added, characterized in that theaforementioned polynucleotide is held on the chromosome and expressessaid potassium channel in somatic cells,

[0011] (3) the transgenic animal described in (1) or (2), wherein it isobtained by effecting ontogenesis of a totipotent cell introduced,together with a promoter, with a polynucleotide encoding a potassiumchannel which is a polypeptide consisting of the amino acid sequencerepresented by SEQ ID NO:2 or a polypeptide which comprising an aminoacid sequence in which from 1 to 10 of amino acids in the amino acidsequence represented by SEQ ID NO:2 are deleted substituted and/or addedand is expressed limitedly to the brain,

[0012] (4) a transgenic animal introduced with a sequence comprising apromoter and a polynucleotide encoding a polypeptide which consists ofan amino acid sequence having a homology of 90% or more with the aminoacid sequence represented by SEQ ID NO:2 and is a potassium channel,

[0013] (5) a transgenic animal which is a non-human animal or aoffspring animal thereof obtained by effecting ontogenesis of atotipotent cell introduced, together with a promoter, with apolynucleotide encoding a polypeptide which consists of an amino acidsequence having a homology of 90% or more with the amino acid sequencerepresented by SEQ ID NO:2 and is a potassium channel, characterized inthat the aforementioned polynucleotide is held on the chromosome andexpresses said potassium channel in somatic cells,

[0014] (6) the transgenic animal described in (4) or (5), wherein it isobtained by effecting ontogenesis of a totipotent cell introduced,together with a promoter, with a polynucleotide encoding a potassiumchannel which is a polypeptide consisting of an amino acid sequencehaving a homology of 90% or more with the amino acid sequencerepresented by SEQ ID NO:2 and is expressed limitedly to the brain,

[0015] (7) the transgenic animal described in (1) to (6), wherein thepromoter is a promoter capable of effecting expression of the potassiumchannel limitedly to the brain,

[0016] (8) the transgenic animal described in (1) to (7), wherein thepromoter is an α-calcium-calmodulin-dependent kinase II promoter,

[0017] (9) the transgenic animal described in (1) to (8), wherein thenon-human animal is a mouse,

[0018] (10), a method for detecting whether or not a substance to betested inhibits said potassium channel, characterized in that itcomprises

[0019] a step of administering a substance to be tested to thetransgenic animal described in (1) to (9), and

[0020] a step of measuring learning and memory abilities,

[0021] (11) a method for detecting whether or not a substance to betested inhibits said potassium channel, characterized in that itcomprises

[0022] a step of administering a substance to be tested to thetransgenic animal described in (1) to (9), and

[0023] a step of measuring anxiety,

[0024] (12) a method for detecting an anti-dementia effect or animproving effect on learning and memory, characterized in that itcomprises

[0025] a step of administering a substance to be tested to thetransgenic animal described in (1) to (9), and

[0026] a step of measuring learning and memory abilities,

[0027] (13) a method for detecting an anxiety inhibiting effect,characterized in that it comprises

[0028] a step of administering a substance to be tested to thetransgenic animal described in (1) to (9), and

[0029] a step of measuring anxiety,

[0030] (14) a method for screening a substance for antidementia or asubstance to improve learning and memory, characterized in that itcomprises

[0031] a step of administering a substance to be tested to thetransgenic animal described in (1) to (9), a step of measuring learningand memory abilities, and

[0032] a step of selecting a substance having an anti-dementia effect oran improving effect on learning and memory,

[0033] (15) a method for screening a substance for antianxiety,characterized in that it comprises

[0034] a step of administering a substance to be tested to thetransgenic animal described in (1) to (9), a step of measuring anxiety,and

[0035] a step of selecting a substance having an anxiety inhibitingeffect,

[0036] (16) a process for producing a pharmaceutical composition forantidementia or for improvement and therapy in learning and memory,characterized in that it comprises a step of carrying out screeningusing the screening method described in (14) and a step of making apharmaceutical preparation using a substance obtained by theaforementioned screening, and

[0037] (17) a process for producing a pharmaceutical composition foranti-anxiety action, characterized in that it comprises

[0038] a step of carrying out screening using the screening methoddescribed in (15) and a step of making a pharmaceutical preparationusing a substance obtained by the aforementioned screening.

[0039] Though there are many reports regarding polynucleotides andpolypeptides having a homology of from 98 to 99% with the BEC1 which canbe used in preparing the transgenic animal of the present invention (WO00/05346, WO 00/09534, WO 99/43696), distinct use has not beenelucidated for each of them. Also, though there are descriptionsregarding transgenic animals in the aforementioned WO 00/05346 and WO99/43696, their illustrative preparation methods are not described andthere are no examples showing their actual preparation and nodescriptions which support functions of said animals. On the other hand,the present inventors have disclosed on the BEC1 in WO 99/37677 inaddition to the aforementioned pamphlets, but without descriptionsregarding transgenic animals.

[0040] That is, the BEC1 transgenic animal, the method for screening asubstance for antidementia, a substance to improve learning and memoryand/or a substance for antianxiety, which uses said model animal, andthe method for producing a pharmaceutical composition for antidementia,for improvement of learning and memory and/or antianxiety, described inthe instant application, are inventions accomplished for the first timeby the present inventors.

[0041] Regarding most of the transgenic animals other than the BEC1counterpart, which have so far been reported on their ability to show abehavioral pattern of impairment in learning and memory, it wasnecessary to add some factors such as aging and the like for thedetection of the disorders (Moran P. M. et al., (1995), Proc. Natl.Acad. Sci. USA, 92, 5341-5345; Hsiao K. et al., (1996), Science, 274,99-102; Arendash G. W. et al., (2001), Brain Res., 891, 42-53), butpreparation of such transgenic animals costs and takes time, and it wasnot practical to conveniently mass-prepare and use a transgenic animalcapable of detecting impairment in learning and memory as a behavioralpattern for use in the screening of a substance for antidementia. Sincethe BEC1 transgenic animal described in the instant application shows abehavior of impairment in learning and memory at a young stage, it canbe prepared conveniently and at a low cost and used in the screening ofa substance for antidementia.

BRIEF DESCRIPTION OF THE DRAWINGS

[0042]FIG. 1 is a graph showing a result of an electrophoresis of PCRproducts obtained by carrying out PCR using a genomic DNA as thetemplate isolated from the tail of an infant mouse in order to identifythe transgenic mouse. N indicate a negative control (mouse genomic DNA,338 bp) and P indicate a positive control (transgene, 245 bp).

[0043]FIG. 2 is a graph showing expressed amounts of BEC1 mRNA in theforebrain and cerebellum of F1 transgenic mice (# 6-2, # 7-3, # 6-5, #7-7 and # 9-5) and a wild type (Wt) mouse.

BEST MODE FOR CARRYING OUT THE INVENTION

[0044] The terms to be used in the present invention are described.

[0045] The term “potassium channel” as used herein means a “potassiumchannel protein”, and the term “BEC1” means a “BEC1 protein”. The term“learning” as used herein means to acquire information from the externalworld, and the term “memory” includes preservation and regeneration ofthe information acquired by the leaning, and the process. The term“learning and memory” shows both concepts of “learning” and “memory”,and the term “memorizing and learning ability” means the ability toacquire, preserve and regenerate the outside information.

[0046] The term “transgene” means a gene for transgenic animalpreparation use, which contains a polynucleotide encoding a promoterregion and a potassium channel.

[0047] The following describes the present invention in detail.

[0048] [1] Polynucleotide Contained in the Transgene for use in thePreparation of the Transgenic Animal of the Present Invention andPolypeptide Encoded by Said Polynucleotide

[0049] The potassium channel polypeptide encoded by the polynucleotidecontained in the transgene for use in the preparation of the transgenicanimal of the present invention includes

[0050] (1) a polypeptide consisting of the amino acid sequencerepresented by SEQ ID NO:2,

[0051] (2) a polypeptide comprising an amino acid sequence in which from1 to 10 of amino acids in the amino acid sequence represented by SEQ IDNO:2 are deleted substituted and/or added and is a potassium channel (tobe referred to as functionally equivalent modified one hereinafter), and

[0052] (3) a polypeptide which consists of an amino acid sequence havinga homology of 90% or more with the amino acid sequence represented bySEQ ID NO:2 and is a potassium channel (to be referred to as homologouspolypeptide hereinafter).

[0053] As the functionally equivalent modified one, “a polypeptide whichcomprising the amino acid sequence represented by SEQ ID NO:2 and is apotassium channel”, “a polypeptide which consists of an amino acidsequence in which from 1 to 10, preferably 1 to 7, more preferably 1 to5, of amino acids in the amino acid sequence represented by SEQ ID NO:2are deleted substituted and/or inserted and is a potassium channel” or“a polypeptide which consists of an amino acid sequence in which from 1to 10, preferably 1 to 7, more preferably 1 to 5, of amino acids in theamino acid sequence represented by SEQ ID NO:2 are deleted substitutedand/or inserted and is a potassium channel which is expressed limitedlyto the brain” is desirable, and the “polypeptide which comprising theamino acid sequence represented by SEQ ID NO:2 and is a potassiumchannel” is more desirable.

[0054] Though the homologous polypeptide is not particularly limited,with the proviso that it consists of an amino acid sequence having ahomology of 90% or more with the amino acid sequence represented by SEQID NO:2 and is a potassium channel, it can consist of an amino acidsequence having a homology of preferably 95% or more, more preferably98% or more, with the amino acid sequence represented by SEQ ID NO:2 andis preferably a potassium channel which is expressed limitedly to thebrain. In this connection, the aforementioned term “homology” as usedherein means a value obtained by using parameters prepared as default byClustal program (Higgins and Sharp, Gene, 73, 237-244, 1998; Thompson etal., Nucleic Acid Res., 22, 4673-4680, 1994) retrieval. Theaforementioned parameters are as follows. As Pairwise AlignmentParameters, K tuple 1 Gap Penalty 3 Window 5 Diagonals Saved 5

[0055] The polypeptide encoded by the polynucleotide contained in thetransgene for use in the preparation of the transgenic animal of thepresent invention has been described in the foregoing, and thepolypeptide consisting of the amino acid sequence represented by SEQ IDNO:2, the functionally equivalent modified body thereof and thehomologous polypeptide thereof are generally referred to as “learningand memory potassium channel”. The “learning and memory potassiumchannel” represents a “learning and memory potassium channel protein”. Aprotein as the polypeptide consisting of the amino acid sequencerepresented by SEQ ID NO:2 is called BEC1.

[0056] In addition, the polynucleotide having a nucleotide sequenceencoding the learning and memory potassium channel may be anypolynucleotide with the proviso that it is a polynucleotide consistingof a nucleotide sequence encoding a polypeptide represented by the aminoacid sequence described in SEQ ID NO:2, a functionally equivalentmodified body thereof or a homologous polypeptide thereof. It ispreferably a polynucleotide consisting of a nucleotide sequence encodingthe amino acid sequence described in SEQ ID NO:2, more preferably thenucleotide sequence described in SEQ ID NO:1.

[0057] [2] Method for Preparing Transgenic Animal Characterized in Thatit Expresses Learning and Memory Potassium Channel

[0058] A polynucleotide encoding a learning and memory potassium channelcontained in a transgene can be obtained in accordance with the methoddescribed in WO 99/37677. For example, the BEC1 gene described in SEQ IDNO:1 can be prepared by a method in which an oligonucleotide issynthesized based on a nucleotide sequence of an optional part of aknown sequence (WO 99/37677), and a human cDNA library is screened usingthis as a probe, or by synthesizing an oligonucleotide which hybridizeswith both termini of the cDNA fragment of interest, and preparing it bythe reverse transcriptase-polymerase chain reaction (RT-PCR method)using this as a primer from mRNA isolated from a human cell. Inaddition, a promoter sequence and an enhancer sequence for controllingexpression of the potassium channel are linked to the transgene. Byselecting this promoter/enhancer sequence, said potassium channel can besystemically expressed and can be selectively expressed in a specifiedtissue. Regarding the promoter, though not particularly limited, thepromoter region of an α-calcium-calmodulin-dependent kinase II(α-CaM-kinase II) gene (Mayford, M. et al. (1990), Proc. Natl. Acad.Sci. USA, 93, 13250-13255), the promoter region of a neuron-specificenolase (Quon, D. et al. (1991), Nature, 352, 239-241), the promoterregion of a Thy-1 gene (Vidal, M. et al. (1990), EMBO J., 9, 833-840) orthe like can be used, the use of the promoter region of anα-calcium-calmodulin-dependent kinase II gene is desirable. Since theα-calcium-calmodulin-dependent kinase II gene is brain-selective andparticularly it is a neuronal gene having high expression selectivity tothe forebrain, a gene of interest can be expressed selectively in thebrain, particularly in the forebrain (cerebral cortex and hippocampus),by the use of said promoter.

[0059] More illustratively, a transgene for use in the preparation ofthe transgenic animal of the present invention can be obtained by themethod described in Example 1. As the transgene for use in thepreparation of the transgenic animal of the present invention, a gene inwhich a BEC1 cDNA having a 5′ intron and poly(A) addition signal islinked to a downstream of the promoter region ofα-calcium-calmodulin-dependent kinase II gene can for example be cited.Though the production method of said transgene is not particularlylimited, a method which uses the polymerase chain reaction (Saiki, R. K.et al. (1988), Science, 239, 487-491; to be referred to as PCRhereinafter) can for example be cited. In addition, the genemanipulation techniques of the present invention can be carried out inaccordance with known methods (e.g., Maniatis, T. et al., “MolecularCloning-A Laboratory Manual”, Cold Spring Harbor Laboratory, NY, 1982).

[0060] Firstly, for the preparation of the promoter region ofα-calcium-calmodulin-dependent kinase II gene, a set of primers, such asSEQ ID NO:3 and SEQ ID NO:4, which can amplify the DNA of the presentinvention are designed based on the information of each nucleotidesequence represented by a sequence (Accession No. AJ222796) registeredin a gene data base GenBank.

[0061] A part of said transgene can be obtained by carrying out PCRusing the thus designed aforementioned primer set and a genomic DNA asthe template. As the genomic DNA, a commercially available product (mfd.by Clontech) can be used, or it can be obtained from an animal bloodsample using a commercially available genomic DNA extraction kit (mfd.by QIAGEN). In addition, α-calcium-calmodulin-dependent kinase IIpromoter region can be obtained by integrating the thus obtained DNAinto an appropriate vector.

[0062] On the other hand, the polynucleotide encoding said potassiumchannel can be obtained in the following manner, it is not limited tothis method and can be obtained also by the method described in WO99/37677.

[0063] A mRNA sample including the one encoding said potassium channelis extracted by a known method from a cell or tissue, for example, thehuman brain, having an ability to produce said potassium channel. As theextraction method, the guanidine thiocyanate hot phenol method, theguanidine thiocyanate-guanidine hydrochloride method and the like can beexemplified, but the guanidine thiocyanate cesium chloride method can becited as a desirable method. The cell or tissue having the ability toproduce said potassium channel can be specified by a northern blottingmethod which uses a gene having a nucleotide sequence encoding saidpotassium channel or a part thereof or a western blotting method whichuses an antibody specific for said potassium channel.

[0064] Purification of the mRNA can be carried out in accordance with aconventional method; for example, it can be purified by effectingadsorption of the mRNA to an oligo(dT) cellulose column and then elutingthe same. As occasion demands, the mRNA can be further fractionated by asucrose density gradient centrifugation or the like method. In addition,a commercially available already extracted mRNA can also be used withoutcarrying out extraction of the mRNA.

[0065] Next, a single-stranded cDNA is synthesized by carrying out areverse transcriptase reaction of the thus purified mRNA in the presenceof a random primer or an oligo(dT) primer. This synthesis can be carriedout by a conventional method. The potassium channel DNA of interest isamplified by carrying out PCR using the thus obtained single-strandedcDNA and two primers sandwiching a partial region of the gene ofinterest. The thus obtained DNA is fractionated by an agarose gelelectrophoresis or the like. As occasion demands, a DNA fragment ofinterest can also be obtained by digesting the aforementioned DNA withrestriction enzymes and the like and ligating the fragments.

[0066] Regarding the transgene for use in the preparation of thetransgenic animal of the present invention, the arrangment order of thepolynucleotides encoding the promoter and the learning and memorypotassium channel is not particularly limited, as long as it contains atleast an arbitrary promoter region and a polynucleotide encoding thelearning and memory potassium channel, and the polynucleotide encodingthe learning and memory potassium channel is arranged in such a mannerthat it is set under control of the promoter activity. As described inExample 1, the transgene for use in the preparation of the transgenicanimal of the present invention can be prepared by transferring theaforementioned promoter region and the learning and memory potassiumchannel gene successively to multi-cloning sites of an appropriatevector. As the aforementioned vector, pUC18 (mfd. by Toyobo) can forexample be cited.

[0067] The transgenic animal of the present invention is notparticularly limited, as long as said transgene is introduced and thelearning and memory potassium channel is over-expressed, but it can beprepared, for example, based on a conventionally known method (e.g.,Animal Biotechnology, 1, 175-84, 1990) except that said transgene isused as the gene to be introduced. Illustratively, it can be preparedbased, for example, on the procedure described in Example 1 which isdescribed later. That is, a transgenic animal of interest can beprepared by introducing the aforementioned transgene into a totipotentcell of a non-human animal, allowing this cell to develop intoindividuals and then selecting an individual in which the transgene isintegrated into the genome of somatic cell. The term “animal” as usedherein means an animal excluding human (namely, non-human animal), andits examples include mammals other than human (e.g., rat, mouse, dog,cat, monkey, pig, cattle, sheep, rabbit, goat, dolphin or horse), birds(e.g., domestic fowl or quail), the amphibia (e.g., frog), the reptiles,insects (e.g., Drosophila) and the like. As the non-human animal,technically it is possible to use all animal species, but a mouse isparticularly desirable, because a large number of inbred strains arecreated and, what is more, fertilized egg culturing, in vitrofertilization and the like techniques are well established. As thetotipotent cell into which a gene is introduced, a fertilized egg orearly stage embryo can be used in the case of mouse. In addition,physical injection (microinjection) of DNA is desirable as the methodfor introducing the gene into a cultured cell, when the productionefficiency of transgenic animal individuals and the transferringefficiency of the transgene to the next generation are taken intoconsideration.

[0068] For example, a vector dissolved in HEPES buffer, phosphatebuffer, physiological saline or the like is injected into a fertilizedegg, and this egg is transplanted into the womb of a host animal treatedwith a hormone (PGF₂α, hCG, estradiol, LH or the like) or, in the caseof a small animal, of an individual treated by a physical stimulation,for inducing into a condition of pseudopregnancy. By feeding this hostanimal and allowing it to carry out delivery, a gene-transferrednon-human animal is obtained. Whether nor not a gene-transferrednon-human animal is obtained can be known by extracting DNA from a partof the body (e.g., tail tip) and verifying the presence of the transgeneby the Southern analysis or PCR. When an individual in which thepresence of the transgene was confirmed is used as the founder, thetransgene is transferred to 50% of its offspring, so that it is possibleto prepare a wild type or mutation type animal efficiently.

[0069] The transgenic animal prepared in this manner is useful for amethod for screening an agent targeted said channel, a method fordetecting an anti-dementia effect or learning and memory improvingeffect, a method for detecting an anxiety inhibitory effect, a methodfor screening a substance for antidementia or a substance to improvelearning and memory, and a method for screening a substance forantianxiety.

[0070] Although the substance to be tested which is used in thescreening method of the present invention is not particularly limited,its examples include commercially available compounds (includingpeptides), various conventionally known compounds (including peptides)registered in Chemical File, a group of compounds obtained by thecombinatorial chemistry techniques (N. K. Terrett, M. Gardner, D. W.Gordon, R. J. Kobylecki and J. Steele, Tetrahedron, 51, 8135-37,(1995)), microbial culture supernatants, natural components derived fromplants and marine organisms, animal tissue extracts, and compounds(including peptides) prepared by chemically or biologically modifyingthe compounds (including peptides) selected by the screening method ofthe present invention.

[0071] [3] Method for Measuring Memorizing and Learning Ability andMethod for Detecting (Screening) Substance to Improve Learning andMemory

[0072] The method for measuring memorizing and learning ability andmethod for detecting (screening) substance to improve learning andmemory are described in the following. By the following methods formeasuring memorizing and learning ability, whether or not a compound tobe tested inhibits the learning and memory potassium channel can bedetected.

[0073] 1. A Method Which Uses a Passive Avoidance Test

[0074] The anti-dementia effect and improvement effect on learning andmemory can be detected through the measurement of the memorizing andlearning ability by a passive avoidance test. The passive avoidance testcan be carried out according to McGaugh J. L. (1966), Science, 153,1351-1358, more illustratively by the method described in Example 5.Each compound to be tested is administered before the start of training(e.g., before 15 to 30 minutes) on the first day of the test. When thelatency to enter the dark box of the test compound-administered group isprolonged in comparison with the vehicle-administered group, it can bejudged that the compound has an anti-dementia effect or a learning andmemory improvement effect. The test compound having an anti-dementiaeffect or a learning and memory improvement effect can be selected as asubstance for antidementia or a substance to improve learning andmemory. In this connection, the vehicle-administered group means a groupto which the vehicle of test compound (e.g., 0.5% methyl cellulosephysiological saline or the like) alone was administered.

[0075] 2. A Method Which Uses a Morris Water Maze Test

[0076] The anti-dementia effect and learning and memory improvementeffect can be detected through the measurement of the memorizing andlearning ability by a method which uses a Morris water maze test. Thiscan be carried out as described in Example 4 in accordance with themethod of Wenk G L et al. (1997), Learning and Memory. In CurrentProtocols in Neuroscience, New York, pp.8.5A. 1-8.5B. 7. Each compoundto be tested is administered before the training (e.g., before 0 to 30minutes) on each day. When the escepe latency to the platform in thetest compound-administered group on and after the 2nd day is shortenedin comparison with the vehicle-administered group, it can be judged thatthe compound has an anti-dementia effect or a learning and memoryimprovement effect. The test compound having an anti-dementia effect ora learning and memory improvement effect can be selected as a substancefor antidementia or a substance to improve learning and memory.

[0077] 3. A Method Which Uses a Sound-Clued Fear Conditioning

[0078] The anti-dementia effect and learning and memory improvementeffect can be detected through the measurement of the memorizing andlearning ability by a method which uses a sound-clued fear conditioning.This can be carried out in accordance with the method of Wehner J M etal. (1997), Nature Genetics, 17, 331-334. A sound stimulus (5 kHz for 10seconds) is produced to a transgenic mouse and a constant currentstimulus (0.5 mA for 1 second) is added immediately thereafter. Eachcompound to be tested is administered 30 minutes before the provision ofsound and electricity stimuli. After 24 hours, the sound stimulus aloneis produced to the mouse, and freezing time of the mouse is measured for5 minutes. When the immovable period of time is prolonged in comparisonwith the vehicle-administered group, it can be judged that the compoundhas an anti-dementia effect or a learning and memory improvement effect.The test compound having an anti-dementia effect or a learning andmemory improvement effect can be selected as-a substance forantidementia or a substance to improve learning and memory.

[0079] 4. A Method Which Uses Object Recognition Task

[0080] The anti-dementia effect and learning and memory improvementeffect can be detected through the measurement of the memorizing andlearning ability by a method which uses an object recognition task. Thiscan be carried out in accordance with the method of Ennaceur A andDelacour J (1988), Behavior Brain Research, 31, 47-59. After standingstill two identical objects inside a 60 cm square field, a transgenicmouse is put into the field and allowed to make a search inside thefield freely for 5 minutes. Each test compound is administered 1 hourbefore putting the mouse into the field. One hour thereafter, one of thetwo objects is replaced by a different new object, and the mouse isagain allowed to make a search inside the field for 5 minutes. Adifference between the searching time for the new object and thesearching time for the old object is used as the index of memoryretention, and when this difference is prolonged in the testcompound-administered group, it can be judged that the compound has ananti-dementia effect or a learning and memory improvement effect. Thetest compound having an anti-dementia effect or a learning and memoryimprovement effect can be selected as a substance for antidementia or asubstance to improve learning and memory.

[0081] 5. A Method Which Uses an Eyeblink Conditioning

[0082] The anti-dementia effect and learning and memory improvementeffect can be detected through the measurement of the memorizing andlearning ability by a method which uses an eyeblink conditioning. Themouse eyeblink conditioning can be carried out in accordance with themethod of Chen L et al. (1996), Journal of Neuroscience, 16, 2829-2838.An electrode is embedded into the eyelid muscle of a transgenic mouse torecord electromyogram of the eyelid muscle. A sound stimulus (5 kHz for2 seconds) is produced to the mouse and an electric stimulus is addedimmediately thereafter from the embedded electrode to induce a blink inthe mouse. The sound-electric stimuli are produced 50 times a day, andthis training is carried out continuously 7 days. Each compound to betested is administered every day 1 hour before the provision of stimuli.The frequency of generation of the eyelid muscle activity after thesound stimulus provision and before the electric stimulus provision isused as the index of the conditioned memory acquisition. When thefrequency of generation of the eyelid muscle activity in the testcompound-administered group exceeds that of the vehicle-administeredgroup on and after the 2nd day of the start of the training, it can bejudged that the compound has an anti-dementia effect or a learning andmemory improvement effect. The test compound having an anti-dementiaeffect or a learning and memory improvement effect can be selected as asubstance for antidementia or a substance to improve learning andmemory.

[0083] 6. A Method Which Uses Long-Term Potentiation (LTP) inHippocampus CA1 as the Index

[0084] The anti-dementia effect and learning and memory improvementeffect can be detected through the measurement of the memorizing andlearning ability by a method which uses long-term potentiation (LTP) inhippocampus CA1 as the index. The LTP in hippocampus CA1 can be carriedout according to Tsien, J Z et al. (1996), Cell, 87, 1327-1338 or Oka Het al. (1999), J. Neurosci. Methods, 93, 61-67 or the like, moreillustratively by the method described in Example 7. Each compound to betested is applied before the tetanus stimulation (e.g., before 15 to 30minutes). When the LTP at the time of the test compound addition isaccelerated in comparison with the case of the vehicle addition, it canbe judged that the compound has an anti-dementia effect or a learningand memory improvement effect. The test compound having an anti-dementiaeffect or a learning and memory improvement effect can be selected as asubstance for antidementia or a substance to improve learning andmemory.

[0085] [4] Measuring Method of Anxiety and Detection Method of Substancefor Antianxiety

[0086] Measuring method of anxiety and detection method of a substancefor antianxiety are exemplified in the following. By the followinganxiety measuring methods, whether or not a compound to be testedinhibits the learning and memory potassium channel can be detected.

[0087] 1. A Method Which Uses a Light/Dark Exploration Test

[0088] The anxiety controlling effect can be detected through themeasurement of anxiety by a method which uses a light/dark explorationtest. This can be carried out as described in Example 6 in accordancewith the method of Crawly J N et al. (1997), Current Protocols inNeuroscience, Wiley, New York. Administration of each compound to betested is carried out 30 minutes before the start of the test. When thetime spent in the light box or number of transitions between the lightand dark box in the test compound-administered group exceeds that of thevehicle-administered group, it can be judged that the compound has ananxiety controlling effect. The test compound having an anxietycontrolling effect can be selected as a substance for antianxiety.

[0089] 2. In addition to this, the anxiety controlling effect can bedetected through the measurement of anxiety by a conflict test (Carliniet al., (1978), Mod. Probl. Pharmacopsychiatry, 13, 80-102), an elevatedplus maze (Handley S L and Mithani S (1984), Naunyn-schmiedeberg'sArchive of Pharmacology, 327, 1-5) and the like. When improvement isfound in the scores by these methods after administration of a compoundto be tested to a transgenic mouse, it can be judged that the compoundhas an anxiety controlling effect. The test compound having an anxietycontrolling effect can be selected as a substance for antianxiety.

[0090] [5] Production Method of Pharmaceutical Composition forAnti-dementia, Learning and Memory Improvement and Treatment orAnti-anxiety

[0091] Medicaments can be obtained using, as the main component, asubstance capable of inhibiting the learning and memory potassiumchannel activity, which is selected by the screening method of thepresent invention. These medicaments are useful as a substance forantidementia, a substance to improve learning and memory or a substancefor antianxiety.

[0092] The pharmaceutical preparation which contains a substance capableof inhibiting the learning and memory potassium channel activity as theactive ingredient can be prepared by using carriers, fillers and/orother additive agents generally used in preparing medicaments, inresponse to the type of the active ingredient. Examples of theadministration include oral administration by tablets, pills, capsules,granules, fine subtilaes, powders, solutions for oral use and the likeand parenteral administration by intravenous, intramuscular or the likeinjections, suppositories, percutaneous preparations, transmucosalpreparations and the like. Particularly in the case of peptides whichare digested in the stomach, intravenous injection or the likeparenteral administration is desirable.

[0093] The solid composition for oral administration according to thepresent invention can be prepared by mixing one or more activesubstances with at least one inert diluent such as lactose, mannitol,glucose, microcrystalline cellulose, hydroxypropylcellulose, starch,polyvinyl pyrrolidone, aluminum magnesium silicate or the like. In theusual way, the aforementioned solid composition may contain otheradditives than the inert diluent, such as a lubricant, a disintegratingagent, a stabilizing agent, a solubilizing agent, a solubilizationassisting agent and the like. If necessary, tablets or pills may becoated with a sugar coating or a film of a gastric or enteric substance.The liquid composition for oral administration includes, for example,emulsions, solutions, suspensions, syrups or elixirs and can contain agenerally used inert diluent such as purified water or ethanol. Inaddition to the inert diluent, the aforementioned liquid composition cancontain other additive agent such as a moistening agent, a suspendingagent, a sweetener, an aromatic or an antiseptic.

[0094] The injections for parenteral administration can include asepticaqueous or non-aqueous solutions, suspensions or emulsions. As adiluent, the water-soluble aqueous solutions or suspensions can includedistilled water for injection, physiological saline or the like. As thediluent of the non-aqueous solutions or suspensions, it can include, forexample, plant oil (e.g., propylene glycol, polyethylene glycol or oliveoil), alcohols (e.g., ethanol), polysorbate 80 or the like. Theaforementioned composition can further contain a moistening agent, anemulsifying agent, a dispersing agent, a stabilizing agent, asolubilizing agent, a solubilization assisting agent, an antiseptic orthe like. The aforementioned composition can be sterilized, for example,by filtration through a bacteria retaining filter, blending of agermicide or irradiation. Alternatively, it can be used by firstlymaking into a sterile solid composition and dissolving it in sterilewater or a sterile solvent for injection prior to its use.

[0095] Dose of the medicament containing, as the active ingredient, asubstance capable of inhibiting the learning and memory potassiumchannel activity, which is selected by the screening method of thepresent invention, can be optionally decided by taking intoconsideration strength of the activity of the active ingredient selectedby the aforementioned screening method, and symptoms, age, sex and thelike of each patient to be treated.

EXAMPLES

[0096] The following describes the present invention in detail based onexamples, but the present invention is not restricted by said examples.In this connection, unless otherwise noted, gene manipulation techniquescan be carried out in accordance with the conventionally known methods(Maniatis, T. et al. (1982): “Molecular Cloning—A Laboratory Manual”,Cold Spring Harbor Laboratory, NY and the like). Also, when commerciallyavailable reagents and kits are used, they can be used in accordancewith the instructions attached to the commercial products.

Example 1 Construction of Transgene for BEC1-Overexpressing TransgenicMouse

[0097] The transgene for production of a transgenic mouse overexpressingBEC1 having the amino acid sequence described in SEQ ID NO:2 comprises agene in which a BEC1 cDNA (SEQ ID NO:1) with a 5′ intron and poly(A)addition signal is linked to a downstream of the promoter region ofα-calcium-calmodulin-dependent kinase II gene.

[0098] The promoter region of α-calcium-calmodulin-dependent kinase IIwas obtained as two fragments having a mutually overlapping region, byPCR using a C57BL/6 mouse genomic DNA as the template. The C57BL/6 mousegenomic DNA was purified from a blood sample of the same mouse using agenomic DNA extraction kit (QIAamp DNA Blood Midi Kit, mfd. by QIAGEN).Primers were designed based on the sequence registered in a gene database GenBank (Accession No. AJ222796). A gene fragment of 4.6 kb wasobtained using an oligonucleotide of the nucleotide sequence representedby SEQ ID NO:3 as the forward primer and using an oligonucleotide of thenucleotide sequence represented by SEQ ID NO:4 as the reverse primer. AnAatII recognition sequence is added to the 5′ terminal side of theaforementioned forward primer. In addition, a gene fragment of 3.7 kbwas obtained using an oligonucleotide of the nucleotide sequencerepresented by SEQ ID NO:5 as the forward primer and using anoligonucleotide of the nucleotide sequence represented by SEQ ID NO:6 asthe reverse primer. A SalI recognition sequence is added to the 5′terminal side of the aforementioned reverse primer. Each PCR was carriedout using a DNA polymerase (Pfu Turbo, mfd. by Stratagene) by employinga thermal denaturation at 99° C. (1 minute) and subsequent repetition of45 cycles each comprising 99° C. (15 seconds), 58° C. (15 seconds) and75° C. (10 minutes), or a thermal denaturation at 95° C. (1 minute) andsubsequent repetition of 40 cycles each comprising 95° C. (15 seconds),62° C. (15 seconds) and 75° C. (8 minutes), and the thus obtained genefragment was cloned into a cloning vector (pCR-XL-TOPO plasmid, mfd. byInvitrogen). An endogenous XmaI recognizing sequence is present in theoverlapping region of the 4.6 kb fragment and 3.7 kb fragment. The 4.6kb fragment was digested with restriction enzymes AatII and XmaI, andthe 3.7 kb fragment was digested with restriction enzymes XmaI and SalI.The thus obtained respective fragments were ligated and cloned into aplasmid pUC18 (mfd. by Toyobo) making use of the AatII and SalIrecognition sequences. The α-calcium-calmodulin-dependent kinase IIpromoter region of interest was obtained by the above operation.

[0099] On the other hand, the BEC1 cDNA (SEQ ID NO:1) was obtained as afragment containing a 5′ intron and poly(A) addition signal by PCR usinga potassium channel expression vector pME-E1 (described in WO 99/37677)as the template. An oligonucleotide of the nucleotide sequencerepresented by SEQ ID NO:7 was designed as the forward primer, and anoligonucleotide comprising the nucleotide sequence represented by SEQ IDNO:8 as the reverse primer, respectively from the upstream sequence of5′ intron and downstream sequence of poly(A) addition signal. A SalIrecognition sequence is added to the aforementioned forward primer, andKpnI and NotI recognizing sequences to the reverse primer. PCR wascarried out using a DNA polymerase (Pfu Turbo, mfd. by Stratagene) byemploying a thermal denaturation at 96° C. (1 minute) and subsequentrepetition of 30 cycles each comprising 96° C. (15 seconds), 60° C. (15seconds) and 75° C. (8 minutes). The thus obtained 3.7 kb fragment wascloned into a cloning vector (pCR-XL-TOPO plasmid, mfd. by Invitrogen).This fragment was subcloned into a plasmid pUC18 (mfd. by Toyobo) makinguse of the SpeI recognition sequence and KpnI recognition sequence, andthe aforementioned α-calcium-calmodulin-dependent kinase II promoterregion was further subcloned into its upstream making use of the AatIIrecognition sequence and SalI recognition sequence. A plasmid (namedpCM-E1 plasmid) having a transgene (12 kb) for use in the preparation ofa BEC1-overexpressing transgenic mouse was finally obtained by the aboveoperation.

Example 2. Preparation and Identification of BEC1 Over-expressionTransgenic Mouse

[0100] The transgene (12 kb) for production. of a BEC1-overexpressingtransgenic mouse was cut out from pCM-E1 using restriction enzymes AatIIand NotI and then isolated and purified. The thus obtained gene wasmicro-injected into 283 fertilized eggs of F1 hybrid mice of C57BL/6 andDBA2 mice, and then the resulting fertilized eggs were transplanted intooviducts of ICR foster mother mice (Hogan, B. et al. (1986),Manipulating the mouse embryo: a laboratory manual, Plainview, N.Y.;Cold Harbor Press). The pregnant mice were allowed to undergospontaneous delivery, and the thus obtained 81 offspring mice weresubjected to the identification of transgenic mice.

[0101] In order to identify transgenic mice, PCR was carried out usinggenomic DNA isolated from the tail of each offspring mouse as thetemplate. The genomic DNA was purified from the tail of each mouse usinga genomic DNA extraction kit (MagExtractor -Genome-, mfd. by Toyobo).When an oligonucleotide of the nucleotide sequence represented by SEQ IDNO:9 is designed as the forward primer, and an oligonucleotide of thenucleotide sequence represented by SEQ ID NO:10 as the reverse primer,from the BEC1 cDNA (SEQ ID NO:1), and PCR is carried out using them, a245 bp fragment is amplified from the transgene, and a 338 bp fragmentcontaining 93 bp intron of mouse BEC1 from the mouse genomic DNA. PCRwas carried out on the thus obtained baby mouse genomic DNA preparationsusing these priers. PCR was carried out using a DNA polymerase(AmpliTaq, mfd. by Roche) by employing a thermal denaturation at 94° C.(1 minute) and subsequent repetition of 35 cycles each comprising 94° C.(15 seconds), 60° C. (15 seconds) and 72° C. (30 seconds). The PCRproducts were subjected to an agarose gel electrophoresis, and DNA wasdetected by ethidium bromide staining (FIG. 1). As a result, it wasidentified that 16 of the 81 baby mice are transgenic mice.

Example 3 Determination of BEC1 mRNA

[0102] In order to confirm that the introduced gene is actuallyfunctioning and BEC1 mRNA is over-expressing, expression of BEC1 mRNA inthe brain of transgenic mouse was analyzed. In order to obtain F1 micefor brain extraction use, 11 animals among the 16 transgenic mice werecrossed with C57BL/6 mice. As a result, transfer of the transgene to F1mice was confirmed in 5 transgenic mice. The forebrain and cerebellumwere sampled from each of the thus obtained F1 transgenic mice(4-week-old) to isolate respective RNA. Each RNA was digested with aDNase (mfd. by Promega) for the purpose of preventing contamination ofgenomic DNA. The number of copies of BEC1 mRNA in the thus obtained RNAwas determined by a real time PCR using PRISM 7700 (mfd. by ABI) and afluorescence reagent SYBR Green (mfd. by Molecular Probe). Asingle-stranded cDNA synthesized from each RNA using a reversetranscriptase-polymerase chain reaction kit (Advantage RT-for-PCR Kit,mfd. by Clontech) was used as the template of the real time PCR. Anoligonucleotide of the nucleotide sequence represented by SEQ ID NO:11was designed as the forward primer, and an oligonucleotide pf thenucleotide sequence represented by SEQ ID NO:12 as the reverse primer,from a sequence common to the transgene, human BEC1, and rat and mouseBEC1.

[0103] As a result of the real time PCR, over-expression offorebrain-selective BEC1 mRNA about 10 times higher than that of wildtype was found in 3 lines (# 6-5, # 7-7 and # 9-5) among the 5 lines oftransgenic mice (FIG. 2). By selecting the line # 9-5, expressed amountsof BEC1 mRNA in respective regions of the brain (cerebral cortex,hippocampus, striatum, hypothalamus, thalamus, mid-brain, brain stem,cerebellum) of wild type mouse were compared with those of thetransgenic mouse. As a result, it was confirmed that the BEC1 mRNAover-expression in the transgenic mouse significant in cerebral cortex,hippocampus and striatum in which the expression was also found in thewild type.

Example 4 Analysis of Learned Behavior of BEC1-overexpressing TransgenicMouse in a Morris Water Maze Test

[0104] In order to analyze action of BEC1 overexpression upon learningbehavior, learning behavior of # 9-5 line transgenic mice and that ofwild type mice in a Morris water maze were compared.

[0105] Male 10-week-old transgenic mice (12 animals) and wild type mice(15 animals) were used. A circular pool of 100 cm in diameter was filledwith water which had been clouded using paints, and a circular platformof 10 cm in diameter was arranged at a position of 5 mm below the water.Room temperature and water temperature at the time of the test was 23°C. Swimming pattern of each mouse put into the pool was recorded andanalyzed by a water maze image analyzer (NIH image, mfd. by O'Hara &CO.), and the escape latency to the platform and the time spent in eachquarter area of the pool were measured. One trial of the training wasfixed to 70 seconds, and the training was carried out 3 trials per dayfor 5 days. The escape latency to the platform on the first day of thetraining was almost the same value in both groups, but the escapelatency was prolonged in the transgenic mice than the wild type mice onand after the 3rd day of the start of the training. On the final day ofthe training, the escape latency to the platform (average value±standarddeviation) became 6.9±1.0 seconds in the wild type and 18.1±6.4 secondsin the transgenic mice, thus showing a statistically significantdifference (p<0.05: two-way layout analysis of variance).

[0106] After completion of the training, each mouse was put for 40seconds into the pool from which the platform had been removed, and timeof the mouse spent in the platform-existed area was measured. As aresult, the spent time of transgenic mice was statisticallysignificantly shorter than that of the wild type (p<0.01: Student's ttest).

[0107] The above results show that learning and memory on the platformposition are reduced in the transgenic mice. By this method which usesBEC1 transgenic mice, a substance for antidementia or a substance toimprove learning and memory can be screened.

Example 5 Analysis of Learned Behavior of BEC1-overexpressing TransgenicMouse in a Passive Avoidance Test

[0108] Female # 9-5 line transgenic mice (6 animals) and wild type mice(8 animals), 8-week-old, were used. Each mouse was put into the lightbox region of a light and dark test apparatus for mice (mfd. by O'Hara &Co.), and a constant voltage stimulus of 60 V for 2 seconds was appliedto the mouse when it entered the dark box. The mouse was again put intothe light box 24 hours thereafter, and the latency to the dark box atthis time was measured.

[0109] As a result, the latency to the dark box of the transgenic micewas 167 seconds (median) which was significantly short compared to the600 seconds (median) of the wild type mice (p<0.05: Wilecoxon rank sumtest). It was shown that the ability to learn the dark box-relatedelectric stimulus is reduced in the transgenic mice. By this methodwhich uses BEC1 transgenic mice, a substance for antidementia or asubstance to improve learning and memory can be screened.

Example 6 Analysis of Anxiety of BEC1-overexpressing Transgenic Mouse byLight and Shade

[0110] In order to analyze the action of BEC1 overexpression uponanimals, a light/dark exploration test was carried out using # 9-5 linetransgenic mice and wild type mice. The light/dark explorationing boxes(mfd. by Muromachi Kikai) comprise a 30 cm square light box and anadjoining dark box of 30 cm×20 cm, and a mouse can freely passes thesetwo boxed through a small hole. Each mouse was put into the explorationboxes for 5 minutes, and the time spent in each box and the number oftransitions between the light and dark box were counted by an infraredsensor. As a result, the number of transitions (average value±standarddeviation) of the wild type was 14.3±1.5, while it was significantlyreduced to 9.4±1.9 in the transgenic mice (p<0.05, Student's t test).Also, the light box staying time was 61.4±4.9 seconds in the wild type,but was 42.4±7.5 seconds in the transgenic mice showing significantreduction (p<0.05, Student's t test). It was shown that anxiety isaccelerated in the transgenic mice. By this method which uses BEC1transgenic mice, a substance for antianxiety can be screened.

Example 7 Analysis of LTP in Hippocampus CA1 of BEC1-overexpressingTransgenic Mouse

[0111] It is considered that LTP, a phenomenon in which synaptictransmission efficiency is increased over a long period of time, is abasic process of learning and memory. In order to analyze the action ofBEC1 overexpression upon LTP, comparison of LTP in the hippocampus CA1of # 9-5 line transgenic mice and wild type mice was carried out.

[0112] Hippocampus slices were prepared from 5 animals of each of thetransgenic mouse and wild type mouse of 3 months of age after birth.Preparation of the hippocampus slices was carried out in accordance withthe method of Edwards, F A et al. (1989), “A thin slice preparation forpatch clamp recordings from neurons of the mammalian central nervoussystem”, Pflegers Arch., 414, 600-612. The extracellular electric fieldpotential was recorded using Multi-Electrode Dish (mfd. by MatsushitaElectric Industrial, Oka H et al., (1999), J. Neurosci. Methods, 93,61-67) on which each slice was arranged. The synaptic response of CA1pyramidal cell was induced by stimulating the Shuffercollateral/comissural fibers —CA1 pathway. In the slice of wild typemouse, tetanus stimulation (100 Hz, 1 second) induced increase of thesynaptic reaction (243±26.9% 45 minutes after the tetanus stimulation).On the other hand, similar stimulation induced only a significantlysmall increase (126±13.6%, p<0.05 by Student's t test) in the slice oftransgenic mouse. It was shown that the hippocampus LTP is inhibited inthe transgenic mouse.

[0113] These results suggest a possibility that BEC1 is causing theimpairment in learning and memory by inhibiting formation of thehippocampus LTP. The hippocampus LTP of this transgenic mouse is usefulas an index of a substance for antidementia or a substance to improvelearning and memory.

Example 8 In Vivo Screening of a Substance for Antidementia or aSubstance to Improve Learning and Memory Using Learning and MemoryPotassium Channel Inhibitory Action as the Mechanism

[0114] In vivo screening of a substance for antidementia or a substanceto improve learning and memory using learning and memory potassiumchannel inhibitory action as the mechanism was carried out by a passiveavoidance test similar to Example 5. Each of the compounds to be testedwas suspended in 0.5% methyl cellulose physiological saline andintraperitoneally administered 30 minutes before or just after theconstant voltage stimulation. The mouse was again put into the light box24 hours thereafter, and the shaded box-entering time at this time wasmeasured. One compound having the effect to prolong the shadedbox-entering time in comparison with the 0.5% methyl cellulosephysiological saline-administered group was obtained. It can be judgedthat this compound has an anti-dementia effect or a learning and memoryimproving effect.

Industrial Applicability

[0115] By the screening method of the present invention, a substanceuseful as a substance for antidementia, a substance to improve learningand memory or a substance for antianxiety can be in vivo screened.

[0116] In vivo screening of a substance for antidementia or a substanceto improve learning and memory, which uses the learning and memorypotassium channel inhibitory action as the mechanism, can be carried outby using the learning and memory abilities of the transgenic animal ofthe present invention as the index, and in vivo screening of a substancefor antianxiety, which uses the learning and memory potassium channelinhibitory action as the mechanism, can be carried out by using anxietyof the same animal as the index. That is, the transgenic animal of thepresent invention can be used as a tool for screening a substance forantidementia, a substance to improve learning and memory or a substancefor antianxiety.

[0117] In addition, a pharmaceutical composition for antidementia,learning and memory improvement use or antianxiety can be produced,using a substance capable of inhibiting the learning and memorypotassium channel activity, which can be obtained by the screeningmethod of the present invention, as the active ingredient, and making itinto a pharmaceutical preparation using a carrier, a filler and/or otheradditive agents.

Sequence Listing Free Text

[0118] Description on the “Artificial Sequence” is described in thenumeral index <223> of the following Sequence Listing. Illustratively,respective nucleotide sequences represented by SEQ ID NOs:3, 6, 7 and 8in the Sequence Listing are artificially synthesized primer sequences.

[0119] Though the present invention has been described in the foregoingbased on specific embodiments, modifications and improvements obvious tothose skilled in the art are included in the scope of the invention.

1 12 1 3252 DNA Homo sapiens CDS (1)..(3252) 1 atg ccg gcc atg cgg ggcctc ctg gcg cct cag aac acc ttc ctg gac 48 Met Pro Ala Met Arg Gly LeuLeu Ala Pro Gln Asn Thr Phe Leu Asp 1 5 10 15 acc atc gct acg cgc ttcgac ggc acg cac agt aac ttc gtg ctg ggc 96 Thr Ile Ala Thr Arg Phe AspGly Thr His Ser Asn Phe Val Leu Gly 20 25 30 aac gcc cag gtg gcg ggg ctcttc ccc gtg gtc tac tgc tct gat ggc 144 Asn Ala Gln Val Ala Gly Leu PhePro Val Val Tyr Cys Ser Asp Gly 35 40 45 ttc tgt gac ctc acg ggc ttc tcccgg gct gag gtc atg cag cgg ggc 192 Phe Cys Asp Leu Thr Gly Phe Ser ArgAla Glu Val Met Gln Arg Gly 50 55 60 tgt gcc tgc tcc ttc ctt tat ggg ccagac acc agt gag ctc gtc cgc 240 Cys Ala Cys Ser Phe Leu Tyr Gly Pro AspThr Ser Glu Leu Val Arg 65 70 75 80 caa cag atc cgc aag gcc ctg gac gagcac aag gag ttc aag gct gag 288 Gln Gln Ile Arg Lys Ala Leu Asp Glu HisLys Glu Phe Lys Ala Glu 85 90 95 ctg atc ctg tac cgg aag agc ggg ctc ccgttc tgg tgt ctc ctg gat 336 Leu Ile Leu Tyr Arg Lys Ser Gly Leu Pro PheTrp Cys Leu Leu Asp 100 105 110 gtg ata ccc ata aag aat gag aaa ggg gaggtg gct ctc ttc cta gtc 384 Val Ile Pro Ile Lys Asn Glu Lys Gly Glu ValAla Leu Phe Leu Val 115 120 125 tct cac aag gac atc agc gaa acc aag aaccga ggg ggc ccc gac aga 432 Ser His Lys Asp Ile Ser Glu Thr Lys Asn ArgGly Gly Pro Asp Arg 130 135 140 tgg aag gag aca ggt ggt ggc cgg cgc cgatat ggc cgg gca cga tcc 480 Trp Lys Glu Thr Gly Gly Gly Arg Arg Arg TyrGly Arg Ala Arg Ser 145 150 155 160 aaa ggc ttc aat gcc aac cgg cgg cggagc cgg gcc gtg ctc tac cac 528 Lys Gly Phe Asn Ala Asn Arg Arg Arg SerArg Ala Val Leu Tyr His 165 170 175 ctg tcc ggg cac ctg cag aag cag cccaag ggc aag cac aag ctc aat 576 Leu Ser Gly His Leu Gln Lys Gln Pro LysGly Lys His Lys Leu Asn 180 185 190 aag ggg gtg ttt ggg gag aaa cca aacttg cct gag tac aaa gta gcc 624 Lys Gly Val Phe Gly Glu Lys Pro Asn LeuPro Glu Tyr Lys Val Ala 195 200 205 gcc atc cgg aag tcg ccc ttc atc ctgttg cac tgt ggg gca ctg aga 672 Ala Ile Arg Lys Ser Pro Phe Ile Leu LeuHis Cys Gly Ala Leu Arg 210 215 220 gcc acc tgg gat ggc ttc atc ctg ctcgcc aca ctc tat gtg gct gtc 720 Ala Thr Trp Asp Gly Phe Ile Leu Leu AlaThr Leu Tyr Val Ala Val 225 230 235 240 act gtg ccc tac agc gtg tgt gtgagc aca gca cgg gag ccc agt gcc 768 Thr Val Pro Tyr Ser Val Cys Val SerThr Ala Arg Glu Pro Ser Ala 245 250 255 gcc cgc ggc ccg ccc agc gtc tgtgac ctg gcc gtg gag gtc ctc ttc 816 Ala Arg Gly Pro Pro Ser Val Cys AspLeu Ala Val Glu Val Leu Phe 260 265 270 atc ctt gac att gtg ctg aat ttccgt acc aca ttc gtg tcc aag tcg 864 Ile Leu Asp Ile Val Leu Asn Phe ArgThr Thr Phe Val Ser Lys Ser 275 280 285 ggc cag gtg gtg ttt gcc cca aagtcc att tgc ctc cac tac gtc acc 912 Gly Gln Val Val Phe Ala Pro Lys SerIle Cys Leu His Tyr Val Thr 290 295 300 acc tgg ttc ctg ctg gat gtc atcgca gcg ctg ccc ttt gac ctg cta 960 Thr Trp Phe Leu Leu Asp Val Ile AlaAla Leu Pro Phe Asp Leu Leu 305 310 315 320 cat gcc ttc aag gtc aac gtgtac ttc ggg gcc cat ctg ctg aag acg 1008 His Ala Phe Lys Val Asn Val TyrPhe Gly Ala His Leu Leu Lys Thr 325 330 335 gtg cgc ctg ctg cgc ctg ctgcgc ctg ctt ccg cgg ctg gac cgg tac 1056 Val Arg Leu Leu Arg Leu Leu ArgLeu Leu Pro Arg Leu Asp Arg Tyr 340 345 350 tcg cag tac agc gcc gtg gtgctg aca ctg ctc atg gcc gtg ttc gcc 1104 Ser Gln Tyr Ser Ala Val Val LeuThr Leu Leu Met Ala Val Phe Ala 355 360 365 ctg ctc gcg cac tgg gtc gcctgc gtc tgg ttt tac att ggc cag cgg 1152 Leu Leu Ala His Trp Val Ala CysVal Trp Phe Tyr Ile Gly Gln Arg 370 375 380 gag atc gag agc agc gaa tccgag ctg cct gag att ggc tgg ctg cag 1200 Glu Ile Glu Ser Ser Glu Ser GluLeu Pro Glu Ile Gly Trp Leu Gln 385 390 395 400 gag ctg gcc cgc cga ctggag act ccc tac tac ctg gtg ggc cgg agg 1248 Glu Leu Ala Arg Arg Leu GluThr Pro Tyr Tyr Leu Val Gly Arg Arg 405 410 415 cca gct gga ggg aac agctcc ggc cag agt gac aac tgc agc agc agc 1296 Pro Ala Gly Gly Asn Ser SerGly Gln Ser Asp Asn Cys Ser Ser Ser 420 425 430 agc gag gcc aac ggg acgggg ctg gag ctg ctg ggc ggc ccg tcg ctg 1344 Ser Glu Ala Asn Gly Thr GlyLeu Glu Leu Leu Gly Gly Pro Ser Leu 435 440 445 cgc agc gcc tac atc acctcc ctc tac ttc gca ctc agc agc ctc acc 1392 Arg Ser Ala Tyr Ile Thr SerLeu Tyr Phe Ala Leu Ser Ser Leu Thr 450 455 460 agc gtg ggc ttc ggc aacgtg tcc gcc aac acg gac acc gag aag atc 1440 Ser Val Gly Phe Gly Asn ValSer Ala Asn Thr Asp Thr Glu Lys Ile 465 470 475 480 ttc tcc atc tgc accatg ctc atc ggc gcc ctg atg cac gcg gtg gtg 1488 Phe Ser Ile Cys Thr MetLeu Ile Gly Ala Leu Met His Ala Val Val 485 490 495 ttt ggg aac gtg acggcc atc atc cag cgc atg tac gcc cgc cgc ttt 1536 Phe Gly Asn Val Thr AlaIle Ile Gln Arg Met Tyr Ala Arg Arg Phe 500 505 510 ctg tac cac agc cgcacg cgc gac ctg cgc gac tac atc cgc atc cac 1584 Leu Tyr His Ser Arg ThrArg Asp Leu Arg Asp Tyr Ile Arg Ile His 515 520 525 cgt atc ccc aag cccctc aag cag cgc atg ctg gag tac ttc cag gcc 1632 Arg Ile Pro Lys Pro LeuLys Gln Arg Met Leu Glu Tyr Phe Gln Ala 530 535 540 acc tgg gcg gtg aacaat ggc atc gac acc acc gag ctg ctg cag agc 1680 Thr Trp Ala Val Asn AsnGly Ile Asp Thr Thr Glu Leu Leu Gln Ser 545 550 555 560 ctc cct gac gagctg cgc gca gac atc gcc atg cac ctg cac aag gag 1728 Leu Pro Asp Glu LeuArg Ala Asp Ile Ala Met His Leu His Lys Glu 565 570 575 gtc ctg cag ctgcca ctg ttt gag gcg gcc agc cgc ggc tgc ctg cgg 1776 Val Leu Gln Leu ProLeu Phe Glu Ala Ala Ser Arg Gly Cys Leu Arg 580 585 590 gca ctg tct ctggcc ctg cgg ccc gcc ttc tgc acg ccg ggc gag tac 1824 Ala Leu Ser Leu AlaLeu Arg Pro Ala Phe Cys Thr Pro Gly Glu Tyr 595 600 605 ctc atc cac caaggc gat gcc ctg cag gcc ctc tac ttt gtc tgc tct 1872 Leu Ile His Gln GlyAsp Ala Leu Gln Ala Leu Tyr Phe Val Cys Ser 610 615 620 ggc tcc atg gaggtg ctc aag ggt ggc acc gtg ctc gcc atc cta ggg 1920 Gly Ser Met Glu ValLeu Lys Gly Gly Thr Val Leu Ala Ile Leu Gly 625 630 635 640 aag ggc gacctg atc ggc tgt gag ctg ccc cgg cgg gag cag gtg gta 1968 Lys Gly Asp LeuIle Gly Cys Glu Leu Pro Arg Arg Glu Gln Val Val 645 650 655 aag gcc aatgcc gac gtg aag ggg ctg acg tac tgc gtc ctg cag tgt 2016 Lys Ala Asn AlaAsp Val Lys Gly Leu Thr Tyr Cys Val Leu Gln Cys 660 665 670 ctg cag ctggct ggc ctg cac gac agc ctt gcg ctg tac ccc gag ttt 2064 Leu Gln Leu AlaGly Leu His Asp Ser Leu Ala Leu Tyr Pro Glu Phe 675 680 685 gcc ccg cgcttc agt cgt ggc ctc cga ggg gag ctc agc tac aac ctg 2112 Ala Pro Arg PheSer Arg Gly Leu Arg Gly Glu Leu Ser Tyr Asn Leu 690 695 700 ggt gct ggggga ggc tct gca gag gtg gac acc agc tcc ctg agc ggc 2160 Gly Ala Gly GlyGly Ser Ala Glu Val Asp Thr Ser Ser Leu Ser Gly 705 710 715 720 gac aatacc ctt atg tcc acg ctg gag gag aag gag aca gat ggg gag 2208 Asp Asn ThrLeu Met Ser Thr Leu Glu Glu Lys Glu Thr Asp Gly Glu 725 730 735 cag ggcccc acg gtc tcc cca gcc cca gct gat gag ccc tcc agc ccc 2256 Gln Gly ProThr Val Ser Pro Ala Pro Ala Asp Glu Pro Ser Ser Pro 740 745 750 ctg ctgtcc cct ggc tgc acc tcc tca tcc tca gct gcc aag ctg cta 2304 Leu Leu SerPro Gly Cys Thr Ser Ser Ser Ser Ala Ala Lys Leu Leu 755 760 765 tcc ccacgt cga aca gca ccc cgg cct cgt cta ggt ggc aga ggg agg 2352 Ser Pro ArgArg Thr Ala Pro Arg Pro Arg Leu Gly Gly Arg Gly Arg 770 775 780 cca ggcagg gca ggg gct ttg aag gct gag gct ggc ccc tct gct ccc 2400 Pro Gly ArgAla Gly Ala Leu Lys Ala Glu Ala Gly Pro Ser Ala Pro 785 790 795 800 ccacgg gcc cta gag ggg cta cgg ctg ccc ccc atg cca tgg aat gtg 2448 Pro ArgAla Leu Glu Gly Leu Arg Leu Pro Pro Met Pro Trp Asn Val 805 810 815 ccccca gat ctg agc ccc agg gta gta gat ggc att gaa gac ggc tgt 2496 Pro ProAsp Leu Ser Pro Arg Val Val Asp Gly Ile Glu Asp Gly Cys 820 825 830 ggctcg gac cag ccc aag ttc tct ttc cgc gtg ggc cag tct ggc ccg 2544 Gly SerAsp Gln Pro Lys Phe Ser Phe Arg Val Gly Gln Ser Gly Pro 835 840 845 gaatgt agc agc agc ccc tcc cct gga cca gag agc ggc ctg ctc act 2592 Glu CysSer Ser Ser Pro Ser Pro Gly Pro Glu Ser Gly Leu Leu Thr 850 855 860 gttccc cat ggg ccc agc gag gca agg aac aca gac aca ctg gac aag 2640 Val ProHis Gly Pro Ser Glu Ala Arg Asn Thr Asp Thr Leu Asp Lys 865 870 875 880ctt cgg cag gcg gtg aca gag ctg tca gag cag gtg ctg cag atg cgg 2688 LeuArg Gln Ala Val Thr Glu Leu Ser Glu Gln Val Leu Gln Met Arg 885 890 895gaa gga ctg cag tca ctt cgc cag gct gtg cag ctt gtc ctg gcg ccc 2736 GluGly Leu Gln Ser Leu Arg Gln Ala Val Gln Leu Val Leu Ala Pro 900 905 910cac agg gag ggt ccg tgc cct cgg gca tcg gga gag ggg ccg tgc cca 2784 HisArg Glu Gly Pro Cys Pro Arg Ala Ser Gly Glu Gly Pro Cys Pro 915 920 925gcc agc acc tcc ggg ctt ctg cag cct ctg tgt gtg gac act ggg gca 2832 AlaSer Thr Ser Gly Leu Leu Gln Pro Leu Cys Val Asp Thr Gly Ala 930 935 940tcc tcc tac tgc ctg cag ccc cca gct ggc tct gtc ttg agt ggg act 2880 SerSer Tyr Cys Leu Gln Pro Pro Ala Gly Ser Val Leu Ser Gly Thr 945 950 955960 tgg ccc cac cct cgt ccg ggg cct cct ccc ctc atg gca ccc tgg ccc 2928Trp Pro His Pro Arg Pro Gly Pro Pro Pro Leu Met Ala Pro Trp Pro 965 970975 tgg ggt ccc cca gcg tct cag agc tcc ccc tgg cct cga gcc aca gct 2976Trp Gly Pro Pro Ala Ser Gln Ser Ser Pro Trp Pro Arg Ala Thr Ala 980 985990 ttc tgg acc tcc acc tca gac tca gag ccc cct gcc tca gga gac ctc 3024Phe Trp Thr Ser Thr Ser Asp Ser Glu Pro Pro Ala Ser Gly Asp Leu 995 10001005 tgc tct gag ccc agc acc cct gcc tcc cct cct cct tct gag gaa 3069Cys Ser Glu Pro Ser Thr Pro Ala Ser Pro Pro Pro Ser Glu Glu 1010 10151020 ggg gct agg act ggg ccc gca gag cct gtg agc cag gct gag gct 3114Gly Ala Arg Thr Gly Pro Ala Glu Pro Val Ser Gln Ala Glu Ala 1025 10301035 acc agc act gga gag ccc cca cca ggg tca ggg ggc ctg gcc ttg 3159Thr Ser Thr Gly Glu Pro Pro Pro Gly Ser Gly Gly Leu Ala Leu 1040 10451050 ccc tgg gac ccc cac agc ctg gag atg gtg ctt att ggc tgc cat 3204Pro Trp Asp Pro His Ser Leu Glu Met Val Leu Ile Gly Cys His 1055 10601065 ggc tct ggc aca gtc cag tgg acc cag gaa gaa ggc aca ggg gtc 3249Gly Ser Gly Thr Val Gln Trp Thr Gln Glu Glu Gly Thr Gly Val 1070 10751080 tga 3252 2 1083 PRT Homo sapiens 2 Met Pro Ala Met Arg Gly Leu LeuAla Pro Gln Asn Thr Phe Leu Asp 1 5 10 15 Thr Ile Ala Thr Arg Phe AspGly Thr His Ser Asn Phe Val Leu Gly 20 25 30 Asn Ala Gln Val Ala Gly LeuPhe Pro Val Val Tyr Cys Ser Asp Gly 35 40 45 Phe Cys Asp Leu Thr Gly PheSer Arg Ala Glu Val Met Gln Arg Gly 50 55 60 Cys Ala Cys Ser Phe Leu TyrGly Pro Asp Thr Ser Glu Leu Val Arg 65 70 75 80 Gln Gln Ile Arg Lys AlaLeu Asp Glu His Lys Glu Phe Lys Ala Glu 85 90 95 Leu Ile Leu Tyr Arg LysSer Gly Leu Pro Phe Trp Cys Leu Leu Asp 100 105 110 Val Ile Pro Ile LysAsn Glu Lys Gly Glu Val Ala Leu Phe Leu Val 115 120 125 Ser His Lys AspIle Ser Glu Thr Lys Asn Arg Gly Gly Pro Asp Arg 130 135 140 Trp Lys GluThr Gly Gly Gly Arg Arg Arg Tyr Gly Arg Ala Arg Ser 145 150 155 160 LysGly Phe Asn Ala Asn Arg Arg Arg Ser Arg Ala Val Leu Tyr His 165 170 175Leu Ser Gly His Leu Gln Lys Gln Pro Lys Gly Lys His Lys Leu Asn 180 185190 Lys Gly Val Phe Gly Glu Lys Pro Asn Leu Pro Glu Tyr Lys Val Ala 195200 205 Ala Ile Arg Lys Ser Pro Phe Ile Leu Leu His Cys Gly Ala Leu Arg210 215 220 Ala Thr Trp Asp Gly Phe Ile Leu Leu Ala Thr Leu Tyr Val AlaVal 225 230 235 240 Thr Val Pro Tyr Ser Val Cys Val Ser Thr Ala Arg GluPro Ser Ala 245 250 255 Ala Arg Gly Pro Pro Ser Val Cys Asp Leu Ala ValGlu Val Leu Phe 260 265 270 Ile Leu Asp Ile Val Leu Asn Phe Arg Thr ThrPhe Val Ser Lys Ser 275 280 285 Gly Gln Val Val Phe Ala Pro Lys Ser IleCys Leu His Tyr Val Thr 290 295 300 Thr Trp Phe Leu Leu Asp Val Ile AlaAla Leu Pro Phe Asp Leu Leu 305 310 315 320 His Ala Phe Lys Val Asn ValTyr Phe Gly Ala His Leu Leu Lys Thr 325 330 335 Val Arg Leu Leu Arg LeuLeu Arg Leu Leu Pro Arg Leu Asp Arg Tyr 340 345 350 Ser Gln Tyr Ser AlaVal Val Leu Thr Leu Leu Met Ala Val Phe Ala 355 360 365 Leu Leu Ala HisTrp Val Ala Cys Val Trp Phe Tyr Ile Gly Gln Arg 370 375 380 Glu Ile GluSer Ser Glu Ser Glu Leu Pro Glu Ile Gly Trp Leu Gln 385 390 395 400 GluLeu Ala Arg Arg Leu Glu Thr Pro Tyr Tyr Leu Val Gly Arg Arg 405 410 415Pro Ala Gly Gly Asn Ser Ser Gly Gln Ser Asp Asn Cys Ser Ser Ser 420 425430 Ser Glu Ala Asn Gly Thr Gly Leu Glu Leu Leu Gly Gly Pro Ser Leu 435440 445 Arg Ser Ala Tyr Ile Thr Ser Leu Tyr Phe Ala Leu Ser Ser Leu Thr450 455 460 Ser Val Gly Phe Gly Asn Val Ser Ala Asn Thr Asp Thr Glu LysIle 465 470 475 480 Phe Ser Ile Cys Thr Met Leu Ile Gly Ala Leu Met HisAla Val Val 485 490 495 Phe Gly Asn Val Thr Ala Ile Ile Gln Arg Met TyrAla Arg Arg Phe 500 505 510 Leu Tyr His Ser Arg Thr Arg Asp Leu Arg AspTyr Ile Arg Ile His 515 520 525 Arg Ile Pro Lys Pro Leu Lys Gln Arg MetLeu Glu Tyr Phe Gln Ala 530 535 540 Thr Trp Ala Val Asn Asn Gly Ile AspThr Thr Glu Leu Leu Gln Ser 545 550 555 560 Leu Pro Asp Glu Leu Arg AlaAsp Ile Ala Met His Leu His Lys Glu 565 570 575 Val Leu Gln Leu Pro LeuPhe Glu Ala Ala Ser Arg Gly Cys Leu Arg 580 585 590 Ala Leu Ser Leu AlaLeu Arg Pro Ala Phe Cys Thr Pro Gly Glu Tyr 595 600 605 Leu Ile His GlnGly Asp Ala Leu Gln Ala Leu Tyr Phe Val Cys Ser 610 615 620 Gly Ser MetGlu Val Leu Lys Gly Gly Thr Val Leu Ala Ile Leu Gly 625 630 635 640 LysGly Asp Leu Ile Gly Cys Glu Leu Pro Arg Arg Glu Gln Val Val 645 650 655Lys Ala Asn Ala Asp Val Lys Gly Leu Thr Tyr Cys Val Leu Gln Cys 660 665670 Leu Gln Leu Ala Gly Leu His Asp Ser Leu Ala Leu Tyr Pro Glu Phe 675680 685 Ala Pro Arg Phe Ser Arg Gly Leu Arg Gly Glu Leu Ser Tyr Asn Leu690 695 700 Gly Ala Gly Gly Gly Ser Ala Glu Val Asp Thr Ser Ser Leu SerGly 705 710 715 720 Asp Asn Thr Leu Met Ser Thr Leu Glu Glu Lys Glu ThrAsp Gly Glu 725 730 735 Gln Gly Pro Thr Val Ser Pro Ala Pro Ala Asp GluPro Ser Ser Pro 740 745 750 Leu Leu Ser Pro Gly Cys Thr Ser Ser Ser SerAla Ala Lys Leu Leu 755 760 765 Ser Pro Arg Arg Thr Ala Pro Arg Pro ArgLeu Gly Gly Arg Gly Arg 770 775 780 Pro Gly Arg Ala Gly Ala Leu Lys AlaGlu Ala Gly Pro Ser Ala Pro 785 790 795 800 Pro Arg Ala Leu Glu Gly LeuArg Leu Pro Pro Met Pro Trp Asn Val 805 810 815 Pro Pro Asp Leu Ser ProArg Val Val Asp Gly Ile Glu Asp Gly Cys 820 825 830 Gly Ser Asp Gln ProLys Phe Ser Phe Arg Val Gly Gln Ser Gly Pro 835 840 845 Glu Cys Ser SerSer Pro Ser Pro Gly Pro Glu Ser Gly Leu Leu Thr 850 855 860 Val Pro HisGly Pro Ser Glu Ala Arg Asn Thr Asp Thr Leu Asp Lys 865 870 875 880 LeuArg Gln Ala Val Thr Glu Leu Ser Glu Gln Val Leu Gln Met Arg 885 890 895Glu Gly Leu Gln Ser Leu Arg Gln Ala Val Gln Leu Val Leu Ala Pro 900 905910 His Arg Glu Gly Pro Cys Pro Arg Ala Ser Gly Glu Gly Pro Cys Pro 915920 925 Ala Ser Thr Ser Gly Leu Leu Gln Pro Leu Cys Val Asp Thr Gly Ala930 935 940 Ser Ser Tyr Cys Leu Gln Pro Pro Ala Gly Ser Val Leu Ser GlyThr 945 950 955 960 Trp Pro His Pro Arg Pro Gly Pro Pro Pro Leu Met AlaPro Trp Pro 965 970 975 Trp Gly Pro Pro Ala Ser Gln Ser Ser Pro Trp ProArg Ala Thr Ala 980 985 990 Phe Trp Thr Ser Thr Ser Asp Ser Glu Pro ProAla Ser Gly Asp Leu 995 1000 1005 Cys Ser Glu Pro Ser Thr Pro Ala SerPro Pro Pro Ser Glu Glu 1010 1015 1020 Gly Ala Arg Thr Gly Pro Ala GluPro Val Ser Gln Ala Glu Ala 1025 1030 1035 Thr Ser Thr Gly Glu Pro ProPro Gly Ser Gly Gly Leu Ala Leu 1040 1045 1050 Pro Trp Asp Pro His SerLeu Glu Met Val Leu Ile Gly Cys His 1055 1060 1065 Gly Ser Gly Thr ValGln Trp Thr Gln Glu Glu Gly Thr Gly Val 1070 1075 1080 3 40 DNAArtificial Sequence Primer 3 attcgacgtc gatctttttt ccgtaaactc aataccaggc40 4 20 DNA Mus sp. 4 gcgggcatca aggagtcaag 20 5 20 DNA Mus sp. 5ctcctgtccc tcccgttgac 20 6 29 DNA Artificial Sequence Primer 6acgcgtcgac ctgcccgtgc tcctgagtg 29 7 29 DNA Artificial Sequence Primer 7acgcgtcgac ccaagctctg aaaaaccag 29 8 36 DNA Artificial Sequence Primer 8ggggtaccgc ggccgcgggg atccagacat gataag 36 9 20 DNA Homo sapiens 9cgaggcaagg aacacagaca 20 10 18 DNA Homo sapiens 10 ggggctgcag gcagtagg18 11 20 DNA Homo sapiens 11 agtcacttcg ccaggctgtg 20 12 18 DNA Homosapiens 12 ggggctgcag gcagtagg 18

1. A transgenic animal introduced with a sequence comprising a promoterand a polynucleotide encoding a potassium channel which is a polypeptideconsisting of the amino acid sequence represented by SEQ ID NO:2 or apolypeptide comprising an amino acid sequence in which from 1 to 10 ofamino acids in the amino acid sequence represented by SEQ ID NO:2 aredeleted, substituted and/or added.
 2. A transgenic animal which is anon-human animal or a offspring animal thereof obtained by effectingontogenesis of a totipotent cell introduced, together with a promoter,with a polynucleotide encoding a potassium channel which is apolypeptide consisting of the amino acid sequence represented by SEQ IDNO:2 or a polypeptide comprising an amino acid sequence in which from 1to 10 of amino acids in the amino acid sequence represented by SEQ IDNO:2 are deleted substituted and/or added, characterized in that theaforementioned polynucleotide is held on the chromosome and expressessaid potassium channel in somatic cells.
 3. The transgenic animaldescribed in claim 1 or claim 2, wherein it is obtained by effectingontogenesis of a totipotent cell introduced, together with a promoter,with a polynucleotide encoding a potassium channel which is apolypeptide consisting of the amino acid sequence represented by SEQ IDNO:2 or a polypeptide which comprising an amino acid sequence in whichfrom 1 to 10 of amino acids in the amino acid sequence represented bySEQ ID NO:2 are deleted substituted and/or added and is expressedlimitedly to the brain.
 4. A transgenic animal introduced with asequence comprising a promoter and a polynucleotide encoding apolypeptide which consists of an amino acid sequence having a homologyof 90% or more with the amino acid sequence represented by SEQ ID NO:2and is a potassium channel.
 5. A transgenic animal which is a non-humananimal or a offspring animal thereof obtained by effecting ontogenesisof a totipotent cell introduced, together with a promoter, with apolynucleotide encoding a polypeptide which consists of an amino acidsequence having a homology of 90% or more with the amino acid sequencerepresented by SEQ ID NO:2 and is a potassium channel, characterized inthat the aforementioned polynucleotide is held on the chromosome andexpresses said potassium channel in somatic cells.
 6. The transgenicanimal described in claim 4 or claim 5, wherein it is obtained byeffecting ontogenesis of a totipotent cell introduced, together with apromoter, with a polynucleotide encoding a potassium channel which is apolypeptide consisting of an amino acid sequence having a homology of90% or more with the amino acid sequence represented by SEQ ID NO:2 andis expressed limitedly to the brain.
 7. The transgenic animal describedin claim 1 to claim 6, wherein the promoter is a promoter capable ofeffecting expression of the potassium channel limitedly to the brain. 8.The transgenic animal described in claim 1 to claim 7, wherein thepromoter is an α-calcium-calmodulin-dependent kinase II promoter.
 9. Thetransgenic animal described in claim 1 to claim 8, wherein the non-humananimal is a mouse.
 10. A method for detecting whether or not a substanceto be tested inhibits said potassium channel, characterized in that itcomprises a step of administering a substance to be tested to thetransgenic animal described in claim 1 to claim 9, and a step ofmeasuring learning and memory abilities.
 11. A method for detectingwhether or not a substance to be tested inhibits said potassium channel,characterized in that it comprises a step of administering a substanceto be tested to the transgenic animal described in claim 1 to claim 9,and a step of measuring anxiety.
 12. A method for detecting ananti-dementia effect or an improving effect on learning and memory,characterized in that it comprises a step of administering a substanceto be tested to the transgenic animal described in claim 1 to claim 9,and a step of measuring learning and memory abilities.
 13. A method fordetecting an anxiety inhibiting effect, characterized in that itcomprises a step of administering a substance to be tested to thetransgenic animal described in claim 1 to claim 9, and a step ofmeasuring anxiety.
 14. A method for screening a substance forantidementia or a substance to improve learning and memory,characterized in that it comprises a step of administering a substanceto be tested to the transgenic animal described in claim 1 to claim 9, astep of measuring learning and memory abilities, and a step of selectinga substance having an anti-dementia effect or an improving effect onlearning and memory.
 15. A method for screening a substance forantianxiety, characterized in that it comprises a step of administeringa substance to be tested to the transgenic animal described in claim 1to claim 9, a step of measuring anxiety, and a step of selecting asubstance having an anxiety inhibiting effect.
 16. A process forproducing a pharmaceutical composition for antidementia or forimprovement and therapy in learning and memory, characterized in that itcomprises a step of carrying out screening using the screening methoddescribed in claim 14 and a step of making a pharmaceutical preparationusing a substance obtained by the aforementioned screening.
 17. Aprocess for producing a pharmaceutical composition for anti-anxietyaction, characterized in that it comprises a step of carrying outscreening using the screening method described in claim 15 and a step ofmaking a pharmaceutical preparation using a substance obtained by theaforementioned screening.